The purpose of this proposal is to continue crystallographic studies on cytochrome P450cam from Pseudomonas putida. We have completed refinement of the substrate-bound enzyme to 1.61 angstrom and of the substrate-free enzyme to 2.2 angstrom. We now wish to determine the crystal structure of P450cam complexed with a series of inhibitors and substrates other than camphor to better understand the structural basis for substrate specificity. In addition, we plan to solve the crystal structure of the mechanistically important oxy-P450-camphor ternary complex at cryogenic temperatures. In each of ese studies, special attention will be directed to crystallographic temperature factors as a means of obtaining functionally important dynamical information. In collaboration with Dr. S. Sligar, we will determine the crystal structure of site-directed P450cam variants. As a parallel study on heme enzyme dynamics, we will explore the effect of ligand binding on the conformational and dynamical properties of cytochrome c peroxidase. Finally, we intend to solve the structure of at least one other P450 to directly test ideas we have developed from the P450cam structure on what factors control substrate specificity. The present proposal will complement our overall research program on heme enzyme structure and function. Specifically, we are working on the crystal structures of the cytochrome c peroxidase- cytochrome c complex, horseradish peroxidase, and Pseudomonas aerguinosa cytochrome oxidase. Each of these has been crystallized and work on the peroxidase-cytochrome c complex is well underway.